Collagenase is a zinc-dependent endoproteinase and is a member of the matrix metalloproteinase (MMP) family of enzymes. The MMPs participate in connective tissue remodeling events and aberrant regulation has been associated with several pathologies. The 2.4 angstrom resolution structure of the inhibited enzyme revealed that, in addition to the catalytic zinc, there is a second zinc ion and a calcium ion which play a major role in stabilizing the tertiary structure of collagenase. Despite scant sequence homology, collagenase shares structural homology with two other endoproteinases, bacterial thermolysin and crayfish astacin. The detailed description of protein-inhibitor interactions present in the structure will aid in the design of compounds that selectively inhibit individual members of the MMP family. Such inhibitors will be useful in examining the function of MMPs in pathological processes.

A continuous assay for pp60c-src tyrosine kinase (srcTK) was developed. A lag in phosphorylation of the peptide RRLIEDAEYAARG was observed that could be eliminated by preincubation with MgATP. The induction time for this lag was dependent upon MgATP and srcTK concentrations. When autophosphorylation was monitored by 32P incorporation from [gamma-32P]ATP, a lag in the time course was also observed. These results demonstrate that autoactivation is an intermolecular process. The electrospray ionization mass spectrum of the enzyme before and after activation demonstrated an increase in the phosphorylation state of the enzyme after incubation with MgATP. The delta 85-N-terminal mutant protein and a full-length G2A pp60c-src mutant, which removes the myristylation site, used in these studies were partially phosphorylated on Y338 and Y530 as isolated. This is the first report of phosphorylation on Y338, but the significance of this site of phosphorylation is unknown. These phosphorylations were insufficient to active the enzyme for transfer of the gamma-phosphoryl of MgATP to the peptides. The unphosphorylated enzyme initially present was converted to a monophosphorylated species upon treatment with MgATP. Y-419 phosphorylation was evident only after treatment with MgATP. These data are consistent with autophosphorylation on Y-419 as predicted. Intermolecular autophosphorylation is consistent with the ability of srcTK to dimerize, which is analogous to activation of receptor tyrosine kinases such as the EGF receptor kinase in response to growth factors. These results indicate that dimerization leading to activation does not require binding to the membrane or a hydrophobic N-terminus in the case of srcTK.

Collagenase is a member of the matrix metalloproteinase (MMP) family of enzymes. Aberrant regulation of this family has been implicated in pathologies such as arthritis and metastasis. Two crystal forms of the catalytic (19-kDa) domain of human fibroblast collagenase have been determined using collagenase complexed with a peptide-based inhibitor (CPLX) as a starting model [Lovejoy et al. (1994) Science 263, 375]. The first crystal form (CF1) contains one molecule in the asymmetric unit and has been determined at 1.9-A resolution with an R factor of 19.8%. The second crystal form (CF2) contains two molecules (A and B) in the asymmetric unit and has been determined at 2.1-A resolution with an R factor of 19.7%. The catalytic domain of collagenase is spherical with an active site cleft that contains a ligated catalytic zinc ion. Collagenase shares some structural homology with the bacterial zinc proteinase, thermolysin [Matthews et al. (1972) Nature, New Biol. 238, 37], and the crayfish digestive peptidase, astacin [Bode et al. (1992) Nature 358, 164]. The amino terminus (Leu 102 to Gly 105) of CF1 and CF2 molecules A and B differs from the conformation found in CPLX by bending away from the molecule and interacting with the active site cleft of symmetry-related molecules. In this alternative conformation, both the mainchain nitrogen and carbonyl oxygen of Leu 102 ligate the symmetry-related catalytic zinc. Although there are structural differences in the active site clefts of CF1, CF2, and CPLX, a number of complex-stabilizing interactions are conserved. The structure of collagenase will be useful for developing compounds that selectively inhibit individual members of the closely related matrix metalloproteinase family.

Poly(ethylene glycol) of various sizes was used as a molecular spacer to separate the cell-targeting ligand, folate, from the surface of poly-L-lysine. The resulting ternary macromolecule (pLys-PEG-folate) was investigated in various formulations for its ability to transfect reporter plasmids into receptor-bearing HeLa and IGROV cell lines. Formulations were optimized with respect to DNA content, +/- charge ratio, and the size and amount of PEG substitution off the pLys backbone. Transfection activity was highest 48 h after sample introduction, and PEG 3400 was determined to be the most favorable spacer size tested. pLys-PEG-folate:DNA transfection was also found to be both concentration dependent and saturable; plus, it was blocked by the addition of excess-free folate, indicative of a specific mechanism of uptake. Transfection activity was virtually identical for complexes formed in 10% serum-supplemented media, deionized water, or Hepes buffer. And, cell viability remained greater than 85% at the highest concentrations of pLys-PEG-folate:DNA complexes tested (4.8 microg/mL pLys 331 000; 12 microg/mL DNA). Taken together, these observations provide evidence that pLys-PEG-folate:DNA complexes are taken up specifically by the folate endocytosis pathway, and that the intramolecular spatial distance of the ligand from the pLys backbone dramatically influences transfection.

A number of quinolones and related antibacterial compounds were screened for activity against calf thymus topoisomerase II by using the P4 unknotting and DNA breakage assays. Several compounds from different structural classes which inhibited DNA unknotting with 50% inhibitory concentrations ranging from 8 to 25 ,ug/ml were identified. Two experimental isothiazoloquinolones from this group, designated A-65281 and A-65282, were also found to induce considerable DNA breakage mediated by calf thymus topoisomerase II, with 32P-end-labeled pBR322 as the substrate. These compounds were nearly as potent as teniposide, with DNA breakage activity evident at concentrations as low as 4 ,ug/ml. However, some differences in DNA cleavage patterns from those with teniposide were evident. These studies have thus identified a new class of agents which have activity against both bacterial and eukaryotic type H topoisomerases. The implications of these data for the selectivity of topoisomerase-directed compounds and the potential toxicity of such compounds developed as antibacterial agents are discussed. topoisomerase II preparations (3,23,25,34,35). A similar pattern of effects has been demonstrated for the quinolone antibacterial agents. Treatment of bacteria with the quinolone prototype, nalidixic acid, results in extensive chromosomal damage (7, 11). Drug-induced DNA breakage can also be catalyzed by purified DNA gyrase (8,10,36). In related studies it was recently demonstrated that additional quinolone-binding sites appear upon the formation of gyrase-DNA complexes and that occupancy of these sites correlates with the occurrence of DNA breakage (30).In general, the agents directed against type II topoisomerases are specific for either bacterial or eukaryotic enzymes. However, in view of the mechanistic similarities and the sequence homologies shared by the bacterial and eukaryotic enzymes, the possibility exists that some agents interact with both classes. Indeed, several antibacterial quinolones which inhibit the P4 DNA-unknotting activity of various eukaryotic type II topoisomerases have been identified (1,15,17). Also, ciprofloxacin and several experimental fluroroquinolones induce topoisomerase II-mediated DNA breakage (1,12,24). These findings raised the possibility that additional classes of compounds that interact with both prokaryotic and eukaryotic enzymes will be identified. Additionally, they underscored the need to carefully assess the cross-reactivity of any topoisomerase-directed antibacterial agent against eukaryotic topoisomerases. Studies to address this important question and their implications for potential quinolone toxicity have been discussed in a recent review (13).We report here the identification of two closely related antibacterial isothiazoloquinolones, designated as A-65281 and A-65282, which are potent inhibitors of the P4-unknotting reaction catalyzed by calf thymus topoisomerase II. Moreover, these compounds are potent inducers of DNA breakage by calf thymus topoisomerase II. These data thus